Rapid and flexible – kinetic or end point mode, at room temperature or 37ºC

Robust – high signal-to-background ratios and excellent Z’- factors

Able to be miniaturized – assays can be run in 96-, 384-, or 1536-well plates

Vivid® Substrates are blocked dyes that yield minimal fluorescence until oxidative cleavage occurs (Figure 1A). Metabolism at either of two potential cleavage sites releases the highly fluorescent product. P450 inhibitors are identified by their ability to prevent the production of the fluorescent metabolite. The three-step Vivid® screening assay (Figure 1B) is a simple mix-and-read protocol that does not require stop reagents to conduct the reaction in kinetic mode.

Figure 1. The Vivid® screening assay. A. The Vivid® Substrate fluoresces when it is oxidized via P450 enzyme activity. B. A simple three step protocol is used to test compounds for their ability to inhibit P450 activity. The test compound is added to a reaction containing P450 and the Vivid® Substrate, and the resulting fluorescence reflects the degree of inhibition of P450 activity.

Vivid® Kits and Substrates are available for a variety of P450 isozymes (Table 1). Each kit includes the isozyme-specific P450 BACULOSOMES® Plus Reagent, a Vivid® Substrate, and an NADPH regeneration system. The excitation and emission wavelengths of cleaved Vivid® Substrates are in the visible region and are subject to little or no fluorescent interference from NADPH or most test compounds.

Table 1. Choose the right Vivid® substrate for your needs. Structures of Vivid® substrates and standards.

P450 activity can be inhibited by solvents commonly used to dissolve test compounds. The following sample data (Table 2) is intended as a guide for the selection and use of organic solvents. We always recommend including a solvent control in your experimental design.

Table 2. Values are given as percent inhibition at the indicated solvent concentration. Values preceded by a “+” indicate an increase in activity. Dashed lines indicate inhibition not detected.

Rapid and flexible – kinetic or end point mode, at room temperature or 37ºC

Robust – high signal-to-background ratios and excellent Z’- factors

Able to be miniaturized – assays can be run in 96-, 384-, or 1536-well plates

Vivid® Substrates are blocked dyes that yield minimal fluorescence until oxidative cleavage occurs (Figure 1A). Metabolism at either of two potential cleavage sites releases the highly fluorescent product. P450 inhibitors are identified by their ability to prevent the production of the fluorescent metabolite. The three-step Vivid® screening assay (Figure 1B) is a simple mix-and-read protocol that does not require stop reagents to conduct the reaction in kinetic mode.

Figure 1. The Vivid® screening assay. A. The Vivid® Substrate fluoresces when it is oxidized via P450 enzyme activity. B. A simple three step protocol is used to test compounds for their ability to inhibit P450 activity. The test compound is added to a reaction containing P450 and the Vivid® Substrate, and the resulting fluorescence reflects the degree of inhibition of P450 activity.

Vivid® Kits and Substrates are available for a variety of P450 isozymes (Table 1). Each kit includes the isozyme-specific P450 BACULOSOMES® Plus Reagent, a Vivid® Substrate, and an NADPH regeneration system. The excitation and emission wavelengths of cleaved Vivid® Substrates are in the visible region and are subject to little or no fluorescent interference from NADPH or most test compounds.

Table 1. Choose the right Vivid® substrate for your needs. Structures of Vivid® substrates and standards.

P450 activity can be inhibited by solvents commonly used to dissolve test compounds. The following sample data (Table 2) is intended as a guide for the selection and use of organic solvents. We always recommend including a solvent control in your experimental design.

Table 2. Values are given as percent inhibition at the indicated solvent concentration. Values preceded by a “+” indicate an increase in activity. Dashed lines indicate inhibition not detected.